Coexistence of a novel NDM-1-encoding MDR plasmid and an IMP-4-encoding IncN-IncU hybrid plasmid in a clinical isolate of Citrobacter freundii BC73

Objectives To investigate the genetic characteristics and transmission mechanism of the NDM-1-, IMP-4-, and SHV-12-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii BC73. Methods C. freundii BC73 was isolated from a urine specimen of a urological patient diagnosed with bladder cancer at a Chinese teaching hospital. Antimicrobial susceptibility testing was carried out using DL-120E susceptibility cards and DL-96A system. Whole genome sequencing (WGS) of the isolate was performed using the Illumina and Oxford Nanopore platforms to analyze the genetic context of drug resistance genes and plasmid characteristics. The phylogenetic tree was constructed and visualized by KSNP3.0 software and iTOL5.0 online database. Results C. freundii isolate BC73 co-carrying blaNDM-1, blaIMP-4 and blaSHV-12 were multidrug-resistant. blaNDM-1 and blaIMP-4 were located on a novel IncFIB-like plasmid, pCFBC1, and an IncN-IncU hybrid plasmid, pCFBC2, respectively. The transferability of blaNDM-1 and blaIMP-4 from C. freundii BC73 to E. coli J53 was successfully demonstrated. The genetic context of the blaNDM-1 and blaIMP-4 genes were ISCR27-groEL-∆groES-cutA-dsbD-trpF-bleMBL-blaNDM-1-∆ISAba125-IS3000 and intI1-blaIMP-4-Kl.pn.13-mobC-IS6100, respectively. Additionally, two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2) were identified and numerous antimicrobial resistance genes were discovered on it. Conclusion To our knowledge, our study represents the first characterization of a ST22 C. freundii isolate co-harboring blaNDM-1, blaIMP-4, and blaSHV-12, obtained from a urine sample. The dissemination of this MDR isolate should be of close concern in future clinical surveillance.


Introduction
Infections due to carbapenemase-producing Enterobacteriaceae (CPE) remain pose a major threat to the public health (Nordmann et al., 2011;Tang et al., 2023).In particular, the co-production of two or three carbapenemases in a single bacterial isolate has become increasingly prevalent over the past 5 years, and resistance has shown an increase compared to the presence of a single gene.Such as in the study by Biez et al., the MICs of imipenem, meropenem and ertapenem in bla NDM-1 -E. coli J53 or bla OXA-48 -E. coli J53 transconjugants (Tc) or bla VIM-1 -E. coli TOP10 transformant (Tf) were significantly lower than the original strain NDM-1-, VIM-1-and OXA-48producing C. freundii 255A1.In another report, the MIC of meropenem in the original strain 112,298 was the same as the highest MIC in the transformants (112298-KPC-TOP10 and 112,298-NDM-TOP10) (Feng et al., 2015;Biez et al., 2022).We should beware of the emergence of such strains.The bla NDM-1 and bla IMP-4 genes, both encoding metallo-beta-lactamases (MBLs) with high carbapenemase activity, enable them to hydrolyze nearly all β-lactams including carbapenems.In recent years, they have been frequently detected in a diverse array of gram-negative bacteria, leading to the occurrence of numerous serious outbreaks (Yong et al., 2009;Dolejska et al., 2016;Xiong et al., 2016;Matsumura et al., 2017;Guducuoglu et al., 2018;Roberts et al., 2020).The simultaneous presence of these two resistance genes in a single strain may result in the emergence of highly drug-resistant variants, presenting a significant challenge for the treatment of infections.
Citrobacter freundii, a member of Enterobacteriaceae family and widely existed in water, soil, and the intestines of both animals and humans, has been identified as an opportunistic pathogen responsible for various infections including urinary, gastrointestinal, respiratory, peritoneal and bloodstream infections (Bodey, 2005).Unfortunately, the indiscriminate use of carbapenems has led to an escalating acquired resistance to antibiotics in C. freundii in recent years.So far, carbapenemases such as KPC-2-, NDM-1-, IMP-4-, OXA-48-and VIM-1-type have been reported in C. freundii, with affected regions including China, India, Spain, France and Italy (Yong et al., 2009;Gaibani et al., 2013;Feng et al., 2015;Lalaoui et al., 2019;Biez et al., 2022).However, the coexistence of NDM-1 and IMP-4 in single C. freundii isolate, along with its characteristics of transmission and resistance, has been rarely documented.
In this study, we identified a ST22 isolate of C. freundii, named BC73, which is the first reported case of co-carrying bla NDM-1 , bla IMP-4 and bla SHV-12 from urine.Upon comprehensive investigation, we discovered that bla NDM-1 and bla IMP-4 were carried by a novel MDR plasmid and an IncN-IncU hybrid plasmid, respectively.Additionally, two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2) harboring multiple resistance genes were identified, which were a potential contribution to the dissemination of multiple drug resistance.

Bacterial isolation and susceptibility testing
A urine specimen was obtained from a hospital patient undergoing examination at the Fifth Clinical Medical College of Henan University of Chinese Medicine (FCMC-HUCM), Zhengzhou, China, in December 2021.The sample were cultured on MacConkey agar (OXOID, Hampshire, United Kingdom) plates supplemented with 2 mg/L meropenem and incubated at 37°C for 18-24 h.Species identification was conducted using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker, Bremen, Germany) and 16S rRNA gene sequencing.In vitro susceptibility test was performed using DL-120E susceptibility cards and DL-96A system (Zhuhai Deere Biological Engineering Co., LTD), which included 25 antibacterial agents as listed in Table 1.The interpretation of results followed the guidelines of the Clinical Laboratory Standards Institute (CLSI 2021;Humphries et al., 2021), with the exceptions of tigecycline and colistin, for which clinical breakpoints were determined according to the U.S. Food and Drug Administration (FDA) (Marchaim et al., 2014) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2022) guidelines 1 , respectively.Ultimately, C. freundii BC73 was confirmed and its details were presented in the subsequent results.

Transferability of plasmids carrying bla NDM-1 and bla IMP-4 , respectively
The bla NDM/IMP -carrying plasmids were visualized through PFGE/ S1 nuclease analysis, followed by southern hybridization, utilizing digoxigenin-labeled bla NDM-1 and bla IMP-4 -specific probes.The conjugation transfer of plasmids was executed by co-culturing with the recipient E. coli J53 at a 1:10 donor-to-recipient ratio, maintained at 25°C (Gou et al., 2020).Transconjugants were selectively cultivated on Mueller-Hinton medium supplemented with sodium azide (150 mg/L) and meropenem (2 mg/L).The confirmation of the selected transconjugants were carried out through PCR experiments.

Whole-genome sequencing and data analysis
Total DNA was extracted utilizing the Tiangen Genomic DNA Extraction Kit (Tiangen, Beijing, China) and subsequently sequenced using the Illumina HiSeq 4,000-PE150 (Illumina, San Diego, United States) and Oxford Nanopore GridION (Nanopore, Oxford, United Kingdom) platforms.De novo assembly of both short reads and long reads was conducted using Unicycler v0.4.8 (Wick et al., 2017), and the genomic sequences were annotated through the NCBI prokaryotic genome annotation pipeline.To identify sequence types (ST) and antimicrobial resistance genes, PubMLST 2 and ResFinder 4.5 3 were employed.Replicon types of plasmid were performed by PlasmidFinder 2.1. 4The conjugation transfer modules in plasmids were predicted by oriTfinder 5 and ICEfinder. 6Virulence factors (VFs) and mobile genetic elements (MGEs) were identified using Additionally, sequence comparisons were executed using BLAST. 9 Comparative maps of the gene environment surrounding bla NDM-1 and bla IMP-4 genes were generated by Easyfig (Sullivan et al., 2011) and the BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011) tool.

Phylogenetic analysis
Genome sequences of 45 available C. freundii isolates were downloaded from the NCBI 10 database, with C. freundii B38 (GCA_001702455.1)selected as the reference genome for comparative analysis.Subsequently, C. freundii BC73 and other C. freundii genomes were analyzed based on core genomic single nucleotide polymorphisms (SNPs) using KSNP3.0(Gardner et al., 2015).Finally, a maximum likelihood tree was generated and visualized by iTOL5.0(Letunic and Bork, 2021).

GenBank accession numbers
The complete genome sequence of C. freundii BC73 has been submitted to GenBank and assigned the accession numbers CP117475-CP117478.

Results
Clinical C. freundii BC73 co-carrying bla NDM-1 and bla IMP-4 Carbapenem-resistant C. freundii BC73 was isolated from a urine specimen of the patient who was hospitalized for urinary tract infection.The patient had a history of bladder cancer and had undergone total cystotomy and abdominal fistula drainage three months prior.Subsequent PCR and sequencing confirmed that the isolate was C. freundii carrying both bla NDM-1 and bla IMP-4 (Supplement 1).
(2) The group II intron Kl.pn.13 immediately downstream of bla IMP-4 was disturbed by ISSen4 only in pIMP-HK1500.(3) A common 3′-conserved segment (qacE∆1-sul1) of class 1 integron In823 in p11219-IMP was absent in others plasmid and IS6100 was inserted at the far-end downstream of bla IMP-4 .(4) There was no IS26 upstream of bla IMP-4 only in p11219-IMP and pCFBC-2 (VR1 of Figure 5B).Additionally, due to the fragmentation and rearrangement of genetic content, different variants of Tn6292 were generated in four plasmids, but the most significant variation was found in pCFBC-2, reflecting in a complex sequence inserted abovementioned (VR2 of Figure 5B).

Discussion
Citrobacter freundii resistant to carbapenems has been gradually observed in patients with hospital-acquired infections (Hammerum et al., 2016;Zhang et al., 2023).However, studies on its transmission mechanisms and resistance characteristics, especially in case involving C. freundii carrying multiple carbapenemase genes, were scarce.In the routine collection of CRE strains, we obtained the C. freundii isolate BC73 co-carrying the carbapenemase genes bla NDM-1 , bla IMP-4 , and ESBLs gene bla SHV-12 .It was isolated from the urine of a 64-year-old patient with urinary tract infections resulting from total cystotomy and abdominal fistula drainage.Carbapenems resistant Citrobacter freundii isolates, especially multidrug-resistant strains, were gradually being found in urinary tract infections, which may cause the extension of infection and the increase of patients' suffering (Prussing et al., 2020;Zhang et al., 2021;Ye et al., 2023).The AST results presented in Table 1 showed that C. freundii BC73 and transconjugant BC73-J53 were resistance to all β-lactams, chloramphenicol, minocycline, azithromycin, and gentamicin antibiotics tested.Conversely, they were susceptible to nitrofurantoin, tigecycline, polymyxin B, and amikacin.The resistant phenotype of C. freundii BC73 was consistent with resistant genotype of it, implying resistance determinants were likely responsible for multiple drug resistance.
Recently, IncX3 plasmids carrying bla NDM have commonly been identified in different species of the Enterobacteriaceae (Ye et al., 2023), implying their significant role in bla NDM transfer.Furthermore, a study from Zhang et al. showed a high transfer frequency were observed in pZY-NDM1, a IncX3 plasmid harboring bla NDM-1 gene (Zhang et al., 2021).Interestingly, we identified a nonseparable plasmid, pCFBC-1, co-carrying bla NDM-1 , bla SHV-12 and bla DHA-1 .In this plasmid, we found three noteworthy features: (1) pCFBC-1 (~131 kb) was a novel large plasmid carrying bla NDM-1 gene.Firstly, compared to the most homologous plasmids in Figure 4A, we speculated that pCFBC1 underwent a genetic recombination and a conserved structure (groEL-∆groES-cutA-dsbD-trpF-ble MBL -bla NDM-1 -∆ISAba125-IS3000) was recombined into pCFBC1.To our knowledge, The results of the inhibition zones summarizing the measurements.this was the first time that the bla NDM-1 gene was present in this type plasmid and the resistance of it may be increased.Second, the most homologous plasmids of pCFBC1 have not been systematically analyzed.Based on the above, we believed that pCFBC1 was a novel plasmid.
(2) After comparison with the PlasmidFinder database, pCFBC1 was unable to obtain the replicon type of the plasmid and was defined as a nonseparable plasmid.According to the replicon type of the plasmid with higher homology to pCFBC1, we named it InFIBlike plasmid.
Horizontal gene transfer (HGT) plays a crucial role in the dissemination of bacterial resistance.The primary vehicle of HGT included plasmids, transposons (Tn), insertion sequences (IS) and integrons (In), which possessed the capability to capture and recombine genes associated with antibiotic resistance, heavy metal resistance and virulence, disseminating them with mobile characteristics (Qiao et al., 2023).Our investigation revealed that C. freundii BC73 successfully transferred bla NDM-1 and bla IMP-4 , along with a carbapenem non-susceptible phenotype, to the recipient E. coli J53.This confirmed the natural horizontal gene transfer characteristic across species for bla NDM-1 and bla IMP-4 .From the gene point of view, pCFBC2 had the complete conjugation transfer system, which further verified its autonomous conjugation transfer ability.According to our experimental validation in Figure 3, although pCFBC1 lacked elements related to conjugation transfer except part of relaxase, bla NDM-1 gene in it can be transferred to the recipient E. coli J53, suggesting that pCFBC1 may have been transferred to the recipient E. coli J53 with the help of pCFBC2.In pCFBC-1, bla NDM-1 was located in a conserved structure: ISCR27-groEL-∆groES-cutA-dsbD-trpFble MBL -bla NDM-1 -∆ISAba125-IS3000, resembling the structures found in pZY-NDM1 and pNDM-Cf7308.This structure was a combination of Tn3000 and Tn125 remnants.Previous studies have described the prototype structures of Tn3000 and Tn125 associated with bla NDM-1 , as vital vehicles for its dissemination, namely IS3000-groEL-groES-cutA-dsbD-trpF-ble MBL -bla NDM-1 -ISAba125-IS3000 and ISAba125-ISCR27-groEL-∆groES-cutA-dsbD-trpF-ble MBL -bla NDM-1 -ISAba125, respectively (Poirel et al., 2012;Campos et al., 2015).Compared with traditional Tn3000 and Tn125, one copy of IS3000 and ISAba125 was absent and another copy of ISAba125 was incomplete in pCFBC-1 (Figure 5A).The genetic background of bla IMP-4 was intI1-bla IMP-4 -Kl.pn.13-mobC-IS6100 (MGE1 of pCFBC2).Compared with the genetic context of bla IMP-4 in other plasmids (VR1 of Figure 5B), we observed The identification of plasmids size using S1-PFGE (left) and southern blot and hybridization (right).pCFBC1 plasmid was between 104.5 kb and 138.9 kb, which was positive for a probe against bla NDM-1 .pCFBC2 plasmid was between 54.7 kb and 76.8 kb, which was positive for a probe against bla IMP-4 .including In809,823,823b,1,377,1,456,1,460,and 1,589, has been reported in Enterobacteriaceae (Lee et al., 2017;Matsumura et al., 2017;Dolejska et al., 2018;Liang et al., 2018;Liu et al., 2021;Zhao et al., 2021).The between In823 and In823b depends on the integrality of the intI1 gene.Furthermore, we identified a large MGE2 situated between two fragments (fipA∆1 and fipA∆2) in pCFBC2.In addition to containing the common Tn6292 (IS2-IS26-qnrS1, ISKpn19 and tnp genes) (VR2 of Figure 5B), one repB gene, two toxin-antitoxin proteins (higA and yafQ), mobile elements (IS5075-∆Tn3-IS26) and a class 1 integron carrying gene cassettes aac(6′)-Ib3 and arr-3 were assembled on it.The interruption of the fipA gene has been reported could promote the accumulation of plasmids in diverse hosts and facilitate the aggregation of mobile elements (Yang et al., 2018), which was a beneficial explanation for the formation of this MGE2.Phylogenetic analysis (Figure 6) was conducted to unveil evolutionary characteristics and homology of C. freundii.The results revealed that C. freundii BC73 clustered with C. freundii MEI002, C. freundii CAV1321, C. freundii 064C1, C. freundii CF8_ST22, C. freundii IDR1800045912-01-00, C. freundii P7699, C. freundii MH17-012 N and C. freundii DY2007.Interestingly, these isolates all belonged to the ST22 C. freundii strain and were distributed in different countries over the span of a decade, which suggested that the ST22 C. freundii strains have disseminated globally and they may be highly clonal.Notably, C. freundii BC73 co-carrying bla NDM-1 and bla IMP-4 and C. freundii DY2007 harboring bla NDM-5 , isolated from dongyang, China in 2020 (Ye et al., 2023), were found to be the most closely related isolates.Previous report has described that the differences between bla NDM-1 and bla NDM-5 were represented by mutations at only two specific sites (88, 154) (Sun et al., 2019).Based on above findings, we proposed a bold hypothesis that C. freundii BC73 likely evolved from DY2007 through vertical propagation.

Conclusion
In this study, we identified and characterized the genome of C. freundii BC73 co-carrying bla NDM-1 , bla IMP-4 and bla SHV-12 from an inpatient with urinary tract infection after bladder cancer surgery.The bla NDM-1 and bla IMP-4 genes were located in a novel MDR plasmid and an IncN-IncU hybrid plasmid (pCFBC1, pCFBC2), respectively.In addition, multiple transposition units (ISs + resistant determinants), especially including two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2), were found on it.The dissemination of NDM-1 and IMP-4-producing C. freundii isolates and ISs + resistant determinants should be of close concern in future clinical surveillance.Gene-environment comparison of bla NDM-1 and bla IMP-4 .(A) Genetic environment of bla NDM-1 on pCFBC1 and related plasmids.The regions with highly similar sequences were marked by light gray.The bla NDM-1 genes were shown by red; mobile elements were drawn by green.(B) In VR1, genetic context of bla IMP-4 on pCFBC2 and related plasmids.The bla IMP-4 genes were shown by red.In VR2, the genetic feature of Tn6292-associated area on pCFBC2 and related plasmids.

FIGURE 3
FIGURE 3 Conjugation transfer test and PCR verification of transconjugants of C. freundii strain BC73 with sodium azide-resistant E. coli strain J53 served as the recipient strain.(A) Conjugation transfer test of C. freundii strain BC73.From left to right are the screening results under the selective pressure of sodium azide and meropenem.The arrow represents the transconjugants.(B&C) Testing the transformation of the strain BC73's genome DNA into the natural state of E. coli J53.One microliter of DNA at a concentration of 100 (B) & 1 (C) micrograms per milliliter was mixed with the BC73 strain and two repetitions were made.(D) PCR verification of transconjugants with sodium azide and meropenem.cdgR gene is a specific primer for E. coli.

FIGURE 5
FIGURE 5 material.The author(s) declare that financial support was received for the research, authorship, and/or publication of this article.This research was funded by joint construction project of Henan Province medical science and technology research plan (LHGJ20220793), the famous doctor support project of Zhengzhou (2021ZW-41), the collaborative innovation project of Zhengzhou (2023XTCX052), the Zhejiang Provincial Natural Science Foundation of China (LY23C180001), the 'Leading Goose'R&D program of Zhejiang Province (2023C03045) and the program of Zhejiang agriculture and rural affairs (2023SN|F058).

TABLE 1
The results of antimicrobial susceptibility testing.

TABLE 2
Overall features of the C. freundii BC73 Genome.